Figure 2. High Pi impairs C2C12 cell differentiation. C2C12 cells were differentiated in DMEM/HG plus 2% HS for 3 days and treated for an additional 24 h with the indicated Pi concentrations. The cells were then collected and processed for the following analyses: (A) Number of nuclei per myotube (MYH staining) (B) Fusion index (C) Myotube length (D) Myotube width (E) Immunoblot analysis of myogenic differentiation markers (MyoD, myogenin, MYH and troponin I) and cell cycle regulators (p21 and cyclin D1) in whole-cell C2C12 lysates. β-actin was used as loading control. (F) RT-PCR analysis of MyoD1, myogenin, MYH, troponin I, p21, and cyclin D1. GAPDH was used as loading control. (G) Cell cycle phase distributions. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.